The ubiquitin proteasome system plays a key role in many human diseases, with proteins such as the breast cancer-specific tumor suppressor BRCA1 (breast cancer-1) and the protein mutated in early onset Parkinson's disease Parkin acting as ubiquitin ligases. However, the elucidation of enzyme-substrate relationships between ubiquitin protein ligases and their targets poses a significant challenge. The goal of this proposal is to develop a new technology that enables rapid and simple identification of the substrates for ubiquitin ligases. My approach is to tag the ubiquitin moieties used by a specific ubiquitin ligase with biotin, which then becomes associated with substrate proteins and allows their facile identification. The tagging is carried out by the bacterial biotin ligase, BirA, which attaches biotin to proteins that contain a biotin-acceptor peptide. Two fusion proteins are constructed: one of a ubiquitin ligase to BirA, and the other of ubiquitin to the biotin-acceptor peptide. In this arrangement, when the biotin-acceptor ubiquitin is brought to the ubiquitin ligase, the attached BirA will biotinylate the ubiquitin, marking it as having been acted on by that specific ubiquitin ligase. Substrate proteins to which the biotinylated ubiquitin is subsequently attached can thus be purified using streptavidin chromatography and identified by mass spectrometry. As a first test of the system, I have constructed three BirA-ubiquitin ligase fusions and the biotin-acceptor ubiquitin in the yeast Saccharomyces cerevisiae, and shown that: 1) all proteins are expressed; and 2) the biotin-acceptor ubiquitin is functional, used by ubiquitin ligases in yeast, and biotinylated in vitro by BirA. Once this technology is established, I plan to expand it to carry out the determination of substrates for all the known ubiquitin ligases in yeast, providing a critically needed proteome-wide view of ubiquitin ligases and their substrates. Ultimately, I would adapt the system for the study of ubiquitin ligases in mammalian tissue culture cells. The determination of substrates for human ubiquitin ligases like BRCA1 and Parkin is currently a daunting task, and this system should speed their discovery and shed light on the etiology of several human diseases. [unreadable] [unreadable] [unreadable]